Secondary active transporters are a class of membrane proteins that utilize pre-existing molecular concentration gradients as an energy source for translocating another substrate, such as a nutrient or a neurotransmitter, against its concentration gradient. They do so by changing conformations so as to form a pathway to the substrate binding site(s) on one or other side of the membrane, in a cycle known as alternating access. Every organism expresses dozens of different secondary transporter proteins, based on a diverse set of different architectures, albeit always with some form of internal structural symmetry. In spite of the unprecedented insights from the recently reported three-dimensional structures, a detailed understanding of the mechanism of each membrane transport protein requires knowledge of its structure in many more conformational states, as well as identification of the binding regions for the substrate or substrates. Studies from our group over the last year have provided such insights into a number of biomedically important transporters responsible for inorganic phosphate, succinate or neurotransmitter uptake, as detailed below. In 2014, we predicted the structural fold of a secondary transporter responsible for sodium-coupled phosphate uptake in the kidney, called NaPi-IIa, by identifying an evolutionary relationship with a a sodium-coupled dicarboxylate transporter known as VcINDY, whose structure was then used as a template for homology modeling. Within that model we had proposed binding sites for the substrates (Fenollar-Ferrer et al, Biophysical Journal, 2014; Fenollar-Ferrer et al, Biophysical J, 2015), but the protein had a rather unusual structure, with a large aqueous extracellular cavity, and the other states required for alternating access were enigmatic. We therefore used our previously-developed repeat-swap modeling approach to predict an alternate conformational state for the template protein, VcINDY. The resultant structural model strongly indicated a two-domain elevator-type conformational mechanism, similar to that previously described for the glutamate transporter family (Reyes et al, Nature 2008; Crisman et al, Proc Natl Acad Sci 2009), a mechanism with significant implications for its interaction with the membrane. To test this striking prediction, our collaborators in the Mindell laboratory here at NINDS, used biochemical and biophysical approaches to probe the predicted state (1). The results of these experiments provided strong support that the elevator-like mechanism of secondary transporters is more common than previously anticipated. We subsequently used the new repeat-swapped model of VcINDY as a template for a new model of NaPi-II, which as expected, also predicts an elevator-like motion, and moreover could be used to aid interpretation of elegant voltage-clamp fluorometry measurements performed by the Forster laboratory (2). Both studies were highlighted in their respective journals as notable contributions (Ryan and Vandenberg, Nature Struct Mol Biol, 2016; Gasnier, Biophys J, 2016). Aside from the identification of the conformational states required for alternating access, a major unresolved question for many secondary active transporters is how they bind and respond to their substrates and, by contrast, how inhibitors interfere with their mechanisms. In particular, a long-standing interest of our laboratory is the manner by which neurotransmitters are recycled into the presynaptic neuron by sodium-driven transporters in the neurotransmitter:sodium symporter (NSS) and excitatory amino acid transporter (EAAT) families. In fact, the details of substrate and inhibitor interactions remain poorly understood in many of those transporters. Even one of the highest affinity NSS inhibitors, the antidepressant paroxetine, has a binding mode that has remained ambiguous even in the light of recent high-resolution crystal structures of its target, serotonin transporter (SERT; Coleman et al, Nature, 2016). We used structure prediction and docking methods to predict the binding of paroxetine to SERT orthologs from three different species (3). Experimental modification of the predicted interactions the group of Satinder Singh at Yale University, revealed components of the binding site that are critical for paroxetine specificity. Our study therefore provides an important step towards the rational design of novel and improved antidepressants. Transport and inhibition of NSS transporters requires binding of sodium ions, which are thought to control the conformation of the protein by an unknown mechanism. In a separate study, in collaboration with the Rudnick lab at Yale University, we provided insights into the sodium effect on conformation in an NSS homolog called LeuT, by molecular dynamics simulations of the protein and of mutated versions thereof (4). Together our results indicate that just one of the two sodium binding sites is critical for trapping the transporter into an inhibitor/substrate-binding orientation, whereas a second sodium has more indirect and smaller effects on the conformation of the protein. Finally, we also helped provide a molecular description of the interaction between EAATs and their substrate, by building a homology model of a human transporter based on the structure of a bacterial homolog. We identified amino acids that differ between the human and bacterial proteins, underlying differences in specificity for glutamate and aspartate (5). The Kanner (Jerusalem) and Fahlke (Hannover) labs tested these hypotheses, and identified a single amino acid group as critical for glutamate specificity in neuronal excitatory uptake. The aforementioned studies contribute to furthering our understanding of neurotransmitter recycling in the brain.